Allergic sensitization to lipocalins reflects asthma morbidity in dog dander sensitized children

Abstract Background Sensitization to dog is an important risk factor for asthma in children, but the clinical relevance of IgE to available dog‐ and furry animal allergen molecules is uncertain. Methods Spirometry, methacholine challenge, fraction of exhaled nitric oxide, nasal challenge with dog extract and questionnaires were performed in 59 dog‐sensitized children (age 10–18 years). Serum IgE to dog‐, cat‐, horse extracts and the allergen molecules Can f 1–6, Fel d 1, Fel d 2, Fel d 4 and Equ c 1 were evaluated. Results Median numbers of positive IgE results to furry animal allergen molecules among children without asthma was 3, with asthma 5.5 and with troublesome asthma 9 (asthma vs. no asthma; p = 0.039; troublesome asthma vs. no asthma; p = 0.009). The odds ratio for asthma if sensitized to any lipocalin was 7.2 (95% confidence Interval: 1.44–35.9). Children with troublesome asthma had higher IgE levels to the lipocalins Can f 2, Can f 4 and Can f 6 compared to the rest of the study population (44 vs. 4.1 kUA/L, p = 0.015; 5.8 vs. 0.9 kUA/L, p = 0.018 and 1.3 vs. 0.7 kUA/L, p = 0.03 respectively). Furthermore, a positive nasal challenge was more common among children with troublesome asthma (83% vs. 36%, p = 0.036). Conclusions Polysensitization to furry animal allergens and lipocalins is associated with asthma in dog‐sensitized children. Children with troublesome asthma have higher IgE levels to several dog lipocalins than other dog sensitized children. Key message Polysensitization to furry animal allergens and high IgE levels to the dog lipocalins Can f 2, Can f 4 and Can f 6 is associated with asthma severity in dog dander sensitized children. Molecular allergy diagnostics may thus help the clinicians to evaluate the impact of allergic sensitization on asthma morbidity.


| INTRODUCTION
Allergic sensitization to dog dander, which has increased in recent decades, is a well-established risk factor for asthma in children. 1,2 However, the detection of serum IgE antibodies (IgE) to dog dander has limitations as the rate of asymptomatic sensitization is high, 3 and the content of allergens in dog dander extracts varies. 4,5 Analysis of IgE to the dog allergen molecules Can f 1, Can f 2, Can f 4 and Can f 6, belonging to the lipocalin family, to Can f 3, the dog serum albumin, and to Can f 5, the prostatic kallikrein, offers improved opportunities to shed light on associations between asthma and allergic sensitization at the molecular allergen level. 6 Molecular allergology has been suggested to be particularly useful in patients with polysensitization 7,8 and/or severe asthma. 9 Population based studies have demonstrated that polysensitization and high-titer sensitization to furry animal allergens are associated with persistent asthma. 1,10 Moreover, we have previously shown that polysensitization to the furry animal allergen molecules lipocalin, kallikrein and secretoglobin is associated with increased bronchial inflammation in children with severe asthma. 11 We have previously investigated associations between a nasal challenge with dog dander extract and sensitization to dog allergen molecules among dog dander sensitized children and found that polysensitization is associated with a positive nasal challenge. 12 The primary objective of this study was to investigate the relative importance of IgE reactivity to all six clinically available dog allergen molecules in relation to asthma among dog dander sensitized children. As poly-sensitization to furry animal allergen molecules has shown to be common in children we also included clinically available cat and horse allergen molecules. 10 To our knowledge, no previous study has included analysis of all known dog lipocalins in relation to asthma. In addition, we investigate sensitization in relation to the severity of asthma, evaluated by asthma control, markers of airway inflammation and bronchial hyperresponsiveness.

| Patient population
Sixty patients aged 10-18 years with confirmed sensitization to dog dander (skin prick test wheal size > 3 mm or serum IgE antibodies to dog dander ≥0.10 kU A /L) were included from pediatric outpatient clinics in the Stockholm area, regardless of history of clinical symptoms to dog. One patient did not complete the examinations and was excluded. The study protocol was approved by the Regional Ethics Committee of Karolinska Institutet, Stockholm (Dnr 2014/1453-31/ 4). Written informed consent was obtained from participants and their parents.
All children and their parents were interviewed regarding clinical history, current symptoms, symptom triggers (exposure to dog, cat and horse, pollen and non-allergic triggers such as infections and exercise) and medications for asthma and rhinitis according to a modified version of the standardized questionnaire employed in the Environmental and Childhood Asthma Study. 13 Asthma control was assessed according to the Pediatric Asthma Control Test (ACT) among children 10-11 years of age (maximum score 27) and ACT for individuals above the age of 12 (maximum score 25). A score below 20 indicates deficient asthma control for both tests. 14,15 Asthma was defined as reported physician's diagnosis of asthma verified by review of medical charts. Troublesome asthma was defined as asthma diagnosis in combination with exhaled fraction of exhaled nitric oxide (FeNO) > 35 ppb, Methacholine PD20 < 2 μmol and ACT score < 20.

| Exhaled nitric oxide, pulmonary function and assessment of bronchial hyper responsiveness
A NIOXTM analyzer (Aerocrine AB) was used to measure the FeNO, in accordance with international guidelines. 16 A FeNO level above 20 ppb was considered elevated and above 35 ppb was considered high. 17 Spirometry was performed using a Vitalograph ® 2120 (Vitalograph ® , Ennis, Ireland), using the reference values reported by Polgar. 18 Bronchial hyperresponsiveness to methacholine was assessed utilizing a Spira nebulizer (Spira Respiratory Care Center, Hämeenlinna, Finland). The dose of inhaled methacholine leading to a 20% drop in FEV1 (PD20) was calculated. 19

| Nasal provocation testing
Nasal provocation testing (NPT) was performed as previously described 12 with a commercially available dog dander extract, Aquagen 100,000 SQ-U/ml (ALK-Abello, Copenhagen, Denmark) according to a modified Lebel protocol. 20

| Blood analysis
Blood samples were taken and the white blood cell count was performed. IgE antibodies against dog-cat-and horse dander extract, and the dog allergen molecules Can f 1, Can f 2, Can f 3, and Can f 5 were analyzed by ImmunoCAP. Can f 4 and Can f 6 were produced as recombinant proteins and analyzed by Streptavidin ImmunoCAP. 21,22 Sera that scored positive (IgE ≥ 0.10 kU A /l) for cat or horse extracts were further analyzed for IgE against allergen molecules from cat (Fel d 1, Fel d 2, Fel d 4) and horse (Equ c 1).
All IgE determinations were analyzed by using the ImmunoCAP System (Phadia AB/Thermo Fisher Scientific) according to the manufacturer's instructions. Results are presented as kilounits of allergen per liter, where the cutoff for allergen specific IgE was ≥0.10 kU A /L. Polysensitization refers to the presence of specific IgE to more than one furry animal allergen molecule.

| Statistics
Categorical data were compared by using the Chi

| Clinical characteristics
Fifty of 59 dog dander sensitized children had asthma. Among the 50 children with asthma, 30 reported asthma triggered by dog exposure of whom six had troublesome asthma. The majority of children with asthma also reported allergic rhinitis (48/50; 96%), of whom 70% (n = 35) reported rhinitis triggered by dog exposure.
Nine of the dog dander sensitized children did not have asthma, but allergic rhinitis only, of whom 5 reported rhinitis triggered by dog.
Children with asthma showed a trend towards reacting more frequently upon NPT with dog dander extract compared to the nonasthmatic children (46% vs. 11%, p = 0.07). Furthermore, asthma diagnosis was associated with a reduced score on the ACT and pronounced bronchial hyperreactivity (Table 1). There were no significant differences in exposure to dogs or cats at home between the groups.   Table 2. Following Bonferroni correction, the association between lipocalin sensitization and asthma as well as troublesome asthma ( Figure 1B) and between sensitization to furry animal allergen molecules and troublesome asthma ( Figure 1A) remained statistically significant, whereas the associations between single allergen molecules and asthma were no longer significant.   (Table E 1, Supporting Information).

No significant associations between sensitization rates or IgE levels
to the investigated allergens and pronounced bronchial hyperreactivity were observed (Table E 3, Supporting Information).

| DISCUSSION
This well characterized cohort of dog dander sensitized children represents an important and common patient group in pediatric clinics. We found that asthma was associated with polysensitization to allergen molecules and to lipocalins from furry animals. In addition, we observed that troublesome asthma was associated with a higher degree of polysensitization, increased IgE levels to the dog lipocalins that the severity of the disease in the upper airways is also reflected in the lower airways. 24 Allergic rhinitis among pre-school children has been associated with airway hyperresponsiveness at the age of seven. 25 As allergic rhinitis often precedes the onset of allergic asthma and may deteriorate asthma symptoms through impaired nasal function, children with allergic rhinitis to dog should be thoroughly treated and further evaluated regarding asthma. 26 The association between asthma and IgE to the dog lipocalins Can f 4 and Can f 6 has not been previously investigated. However, the lipocalin protein group has been pointed out as one of two protein families associated with asthma in children, 27 and sensitization to three or more lipocalins has previously been associated with severe asthma. 28 As IgE reactivity to Can f 6 was associated with asthma and IgE levels to Can f 4 and Can f 6 were higher among children with troublesome asthma, we suggest that these allergens could be regarded as markers for asthma among dog dander sensitized children.
In the present study, a vast majority of the children was also sensitized to allergen molecules from cat and horse, and the most common sensitizing allergen was the major cat allergen secretoglobin, Fel d 1. The IgE levels to Fel d 1 did not differ significantly between children with and without asthma or with different asthma manifestations. It has previously been reported that sensitization to Fel d 1 alone is a poor predictor of asthma in children, unless at high titers or in combination with the cat lipocalin Fel d 4. 10 Asthma is a multi-factorial disease. In children, the chronic inflammation is often to a large extent caused by allergic sensitization, while the bronchial hyperreactivity may depend on a broader range of factors. 29 This may explain why we observed elevated IgE levels to dog lipocalins in children with high exhaled FeNO levels as well as insufficient asthma control, while no such associations were seen among children with bronchial hyperreactivity. 30 The major strength of this study is that, for the first time, all clinically available dog allergen molecules were evaluated by a quantitative measure in patients that were extensively investigated regarding asthma manifestations. We also performed nasal provocation tests to elucidate associations between dog induced rhinitis and asthma, and not only sensitization patterns and asthma. We are aware of power limitations due to small sub-groups and risk of significant findings by chance due to multiple testing. Adjusting for T A B L E 2 Sensitization rates and IgE levels to furry animal allergen extracts and molecules among dog dander sensitized children with and without asthma Asarnoj report personal fees from Meda/Mylan, Orion Pharma, ALK F I G U R E 3 IgE levels among sensitized children with troublesome asthma "ABC" (n = 6), with pronounced bronchial hyperreactivity (PD20 < 2 μmol), poor asthma control (Asthma Control Test (ACT) < 20) and high levels of exhaled fraction of exhaled nitric oxide (FeNO) (>35 ppb) compared with IgE levels among sensitized children with controlled asthma or rhinitis only, "others" (n = 51). Horizontal bars indicate median levels. Levels above 100 kU A /L were set at 100 kU A /L KÄCK ET AL.